The IL-2 receptor promotes lymphocyte proliferation and induction of the c-myc, bcl-2, and bcl-x genes through the trans-activation domain of Stat5

Studies assessing the role of Stat5 in the IL-2 proliferative signal have produced contradictory, and thus inconclusive, results. One factor confounding many of these studies is the ability of IL-2R to deliver redundant mitogenic signals from different cytoplasmic tyrosines on the IL-2R beta-chain (IL-2Rbeta). Therefore, to assess the role of Stat5 in mitogenic signaling independent of any redundant signals, all cytoplasmic tyrosines were deleted from IL-2Rbeta except for Tyr510, the most potent Stat5-activating site. This deletion mutant retained the ability to induce Stat5 activation and proliferation in the T cell line CTLL-2 and the pro-B cell line BA/F3. A set of point mutations at or near Tyr510 that variably compromised Stat5 activation also compromised the proliferative signal and revealed a quantitative correlation between the magnitude of Stat5 activation and proliferation. Proliferative signaling by a receptor mutant with a weak Stat5 activating site could be rescued by overexpression of wt Stat5a or b. Additionally, the ability of this receptor mutant to induce c-myc, bcl-x, and bcl-2 was enhanced by overexpression of wt Stat5. By contrast, overexpression of a version of Stat5a lacking the C-terminal trans-activation domain inhibited the induction of these genes and cell proliferation. Thus, Stat5 is a critical component of the proliferative signal from Tyr510 of the IL-2R and regulates expression of both mitogenic and survival genes through its trans-activation domain.     

Functional regions of the mouse interleukin-10 receptor cytoplasmic domain.

The functions of wild-type and mutant mouse interleukin-10 receptors (mIL-10R) expressed in murine Ba/F3 cells were studied. As observed previously, IL-10 stimulates proliferation of IL-10R-expressing Ba/F3 cells. Accumulation of viable cells in the proliferation assay is to a significant extent balanced by concomitant cell death. Moreover, growth in IL-10 also induces a previously unrecognized response, differentiation of the cells, as evidenced both by formation of large clusters of cells in cultures with IL-10 and by induction or enhancement of expression of several cell surface antigens, including CD32/16, CD2, LECAM-1 (v-selectin), and heat-stable antigen. Two distinct functional regions near the C terminus of the mIL-10R cytoplasmic domain which mediate proliferation were identified; one of these regions also mediates the differentiation response. A third region proximal to the transmembrane domain was identified; removal of this region renders the cell 10- to 100-fold more sensitive to IL-10 in the proliferation assay. In cells expressing both wild-type and mutant IL-10R, stimulation with IL-10 leads to tyrosine phosphorylation of the kinases JAK1 and TYK2 but not JAK2 or JAK3 under the conditions tested.     

Abnormal Th1 cell differentiation and IFN-gamma production in T lymphocytes from motheaten viable mice mutant for Src homology 2 domain-containing protein tyrosine phosphatase-1

Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) plays an important role in T and B lymphocyte signaling; however, the function of SHP-1 in Th cell differentiation, in particular, the Th1 response, has not been defined. In this study, we provide evidence that SHP-1 phosphatase negatively regulates Th1 cell development and IFN-gamma production. Compared with the wild-type control, anti-CD3-activated mouse T lymphocytes carrying the motheaten viable mutation in the SHP-1 gene produced a significantly increased amount of IFN-gamma in the presence of IL-12. This increase was also seen at the basal level without IL-12 addition. Similarly, Th1 cell differentiation and proliferation of anti-CD3-activated SHP-1 mutant lymph node cells in the presence or absence of IL-12 were markedly enhanced, indicating a negative role for SHP-1 phosphatase in such lymphocyte activities. Interestingly, IL-12-induced activation of Jak2 and STAT4, critical components for IL-12-mediated cellular responses, was shortened or attenuated in mutant T cells. Together these results suggest that SHP-1 negatively regulates Th1 cell development and functions through a mechanism that is not directly related to IL-12 signaling.     

Cutting edge: TCR stimulation by antibody and bacterial superantigen induces Stat3 activation in human T cells.

Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2R alpha promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways.     

Control of T cell development in vivo by subdomains within the IL-7 receptor alpha-chain cytoplasmic tail

IL-7/IL-7R signaling functions in both growth and differentiation during T cell development. In this study, we examined the extent these activities were controlled by signaling associated with distinct IL-7R alpha cytoplasmic domains by transgenic expression of wild-type or cytoplasmic deletion mutants of IL-7R alpha in the thymi of IL-7R alpha(-/-) mice. We show an essential requirement for the tyrosine-containing carboxyl-terminal T domain in restoring thymic cellularity, pro-/pre-T cell progression, and survival. In contrast, the functional differentiation of TCR alpha beta cells and the development of TCR gamma delta cells are partially independent of the T domain. Thus, separate cytoplasmic domains of the IL-7R alpha chain differentially control distinct functions during T cell development, whereas normal IL-7R-dependent thymic development requires the integrated activity of all these domains.     

Defective T-cell activation is associated with augmented transforming growth factor Beta sensitivity in mice with mutations in the Sno gene

The proto-oncogene Sno has been shown to be a negative regulator of transforming growth factor beta (TGF-beta) signaling in vitro, using overexpression and artificial reporter systems. To examine Sno function in vivo, we made two targeted deletions at the Sno locus: a 5' deletion, with reduced Sno protein (hypomorph), and an exon 1 deletion removing half the protein coding sequence, in which Sno protein is undetectable in homozygotes (null). Homozygous Sno hypomorph and null mutant mice are viable without gross developmental defects. We found that Sno mRNA is constitutively expressed in normal thymocytes and splenic T cells, with increased expression 1 h following T-cell receptor ligation. Although thymocyte and splenic T-cell populations appeared normal in mutant mice, T-cell proliferation in response to activating stimuli was defective in both mutant strains. This defect could be reversed by incubation with either anti-TGF-beta antibodies or exogenous interleukin-2 (IL-2). Together, these findings suggest that Sno-dependent suppression of TGF-beta signaling is required for upregulation of growth factor production and normal T-cell proliferation following receptor ligation. Indeed, both IL-2 and IL-4 levels are reduced in response to anti-CD3 epsilon stimulation of mutant T cells, and transfected Sno activated an IL-2 reporter system in non-T cells. Mutant mouse embryo fibroblasts also exhibited a reduced cell proliferation rate that could be reversed by administration of anti-TGF-beta. Our data provide strong evidence that Sno is a significant negative regulator of antiproliferative TGF-beta signaling in both T cells and other cell types in vivo.     

Stat3-dependent induction of p19INK4D by IL-10 contributes to inhibition of macrophage proliferation

We have previously reported that IL-10 inhibits proliferation of normal bone marrow-derived macrophages and of the monocyte/macrophage cell line J774. Activation of Stat3 was shown to be necessary and sufficient to mediate inhibition of proliferation. To investigate further the mechanism of growth arrest, we examined the effect of IL-10 on expression of cell cycle inhibitors. We found that IL-10 treatment increases expression of the cyclin-dependent kinase inhibitors p19INK4D and p21CIP1 in macrophages. IL-10 cannot induce p19INK4D expression or block proliferation when Stat3 signaling is blocked by a dominant negative Stat3 or a mutant IL-10Ralpha which does not recruit Stat3 in J774 cells, whereas p21CIP1 induction is not affected. An inducibly active Stat3 (coumermycin-dimerizable Stat3-Gyrase B), which suppresses J774 cell proliferation, also induced p19INK4D expression. Sequencing of the murine p19INK4D promoter revealed two candidate Stat3 binding sites, and IL-10 treatment activated a reporter gene controlled by this promoter. These data suggest that Stat3-dependent induction of p19INK4D mediates inhibition of proliferation. Enforced expression of murine p19INK4D cDNA J774 cells significantly reduced their proliferation. Use of antisense p19INK4D and analysis of p19INK4D-deficient macrophages confirmed that p19INK4D is required for optimal inhibition of proliferation by IL-10, and indicated that additional IL-10 signaling events contribute to this response. These data indicate that Stat3-dependent induction of p19INK4D and Stat3-independent induction of p21CIP1 are important components of the mechanism by which IL-10 blocks proliferation in macrophages.     

Characterization of the roles of TNF receptor-associated factor 6 in CD40-mediated B lymphocyte effector functions

Signaling through CD40 in B cells leads to B cell proliferation, Ig and IL-6 secretion, isotype switching, and up-regulation of surface molecules. TNF receptor-associated factor (TRAF) proteins associate with the cytoplasmic tail of CD40 and act as adapter molecules. Of the six TRAFs identified to date, TRAFs 2, 3, 5, and 6 are reported to associate directly with the cytoplasmic tail of CD40, but previous studies have principally examined transient overexpression of TRAF6 in cells that do not normally express CD40. Thus, we examined the role of TRAF6 in CD40-mediated B lymphocyte effector functions using two approaches. We produced and stably expressed in mouse B cell lines a human CD40 molecule with two cytoplasmic domain point mutations (hCD40EEAA); this mutant fails to bind TRAF6, while showing normal association with TRAFs 2 and 3. We also inducibly expressed in B cells a transfected "dominant-negative" TRAF6 molecule which contains only the C-terminal TRAF-binding domain of TRAF6. Using both molecules, we found that TRAF6 association with CD40 is important for CD40-induced IL-6 and Ig secretion, and that TRAF6 mediates its effects on CD40-stimulated Ig secretion principally through its effects on IL-6 production by the B cell. TRAF6 association with CD40 was also found to be important for B7-1 up-regulation, but not for up-regulation of other surface molecules. Interestingly, however, although we could show TRAF6-dependent CD40-mediated activation of NF-kappaB in 293 kidney epithelial cells, no such effect was seen in B cells, suggesting that TRAF6 has cell-type-specific functions.     

STAT3 is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production

IL-27, a member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in augmentation of Th1 differentiation and suppression of proinflammatory cytokine production. In the present study, we investigated the role of STAT3 in the IL-27-mediated immune functions. IL-27 induced phosphorylation of STAT1, -2, -3 and -5 in wild-type naive CD4+ T cells, but failed to induce that of STAT3 and STAT5 in STAT3-deficient cohorts. IL-27 induced not only proinflammatory responses including up-regulation of ICAM-1, T-box expressed in T cells, and IL-12Rbeta2 and Th1 differentiation, but also anti-inflammatory responses including suppression of proinflammatory cytokine production such as IL-2, IL-4, and IL-13 even in STAT3-deficient naive CD4+ T cells. In contrast, IL-27 augmented c-Myc and Pim-1 expression and induced cell proliferation in wild-type naive CD4+ T cells but not in STAT3-deficient cohorts. Moreover, IL-27 failed to activate STAT3, augment c-Myc and Pim-1 expression, and induce cell proliferation in pro-B BaF/3 transfectants expressing mutant gp130, in which the putative STAT3-binding four Tyr residues in the YXXQ motif of the cytoplasmic region was replaced by Phe. These results suggest that STAT3 is activated through gp130 by IL-27 and is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production. Thus, IL-27 may be a cytokine, which activates both STAT1 and STAT3 through distinct receptor subunits, WSX-1 and gp130, respectively, to mediate its individual immune functions.     

Dok-1 is a positive regulator of IL-4 signalling and IgE response

Interleukin-4 (IL-4) plays an essential role in the control of humoral immunity by regulating lymphocyte proliferation and differentiation, including the T helper type 2 lineage commitment of CD4(+) T cells as well as the isotype switching to IgE in B cells. The adaptor protein Dok-1 is known to have an essential role in the negative regulation of a variety of cytokine signalling events. However, here we have found that the loss of Dok-1 impaired the proliferative response of CD4(+) T cells and B cells to IL-4. Conversely, the forced expression of Dok-1 in the myeloid cell line 32D augmented the IL-4-induced proliferation, indicating a positive role for Dok-1. Tyrosine phosphorylation, and thereby the activation of Stat6 and IRS-2, is critical for IL-4 signalling; however, only the activation of Stat6, not the IRS-2-dependent phosphorylation of Akt, was perturbed in Dok-1-deficient cells stimulated with IL-4. Furthermore, mice lacking Dok-1 showed an impaired IgE response to thymus-dependent antigen. Thus, Dok-1 is a positive regulator of IL-4 signalling and IgE response.     

Stat6-dependent and -independent pathways for IL-4 production

Stat6 has been shown to have a crucial role in the IL-4-dependent differentiation of Th2 cells. In this report, we explore whether in vitro Th2 differentiation driven by altered costimulatory signals or Ag dose is Stat6 dependent. We find that blocking B7-1 signaling in vitro promotes the differentiation of IL-4-secreting Th2 cells in wild-type but not Stat6-deficient T cell cultures. Additionally, stimulation with peptide Ag doses that normally result in the production of Th2 cells in vitro fails to do so in cultures of Stat6-deficient cells. We also demonstrate that Stat6 is required for the in vitro differentiation of CD8+ T cells into IL-4-secreting cytotoxic T cell type 2 cells. However, IL-4 expression is not absolutely dependent on Stat6. We demonstrate that populations of T cells that do not require IL-4 for their development, such as NK T cells, are still competent to secrete IL-4 in the absence of Stat6. These results demonstrate that Stat6 is required for the differentiation program leading to the generation of Th2 and cytotoxic T cell type 2 cells but not for IL-4 expression in cells that do not undergo differentiation in response to IL-4.     

Stat6 and IRS-2 Cooperate in Interleukin 4 (IL-4)-Induced Proliferation and Differentiation but Are Dispensable for IL-4-Dependent Rescue from Apoptosis

Stat6 and IRS-2 are two important signaling proteins that associate with the cytoplasmic tail of the interleukin 4 (IL-4) receptor. Data from numerous in vitro experiments have led to a model for IL-4 signal transduction in which the Stat6 signaling pathway is responsible for the IL-4 induced changes in gene expression and differentiation events, while the IRS-2 signaling pathway provides mitogenic and antiapoptotic signals. In order to determine the relative contributions of these signaling molecules in primary lymphocytes, we have examined IL-4 responses in T cells from mice deficient for either Stat6 or IRS-2 as well as from mice doubly deficient for both genes. Both IRS-2 and, especially, Stat6 are shown to be critically involved in IL-4-induced proliferation of T cells, presumably through the cooperative regulation of the Cdk inhibitor p27kip1. Like Stat6-deficient Th cells, IRS-2-deficient cells are also compromised in their ability to secrete Th2 cytokines, revealing a previously unrecognized role for IRS-2 in Th2 cell development. Although Stat6 and/or IRS-2 expression is required for IL-4-induced proliferative and differentiative responses, both signaling proteins are dispensable for the antiapoptotic effect of IL-4. However, treatment of lymphocytes with a protein tyrosine phosphatase inhibitor is able to block the antiapoptotic effect of IL-4 specifically in Stat6- or IRS-2-deficient cells and not in wild-type cells. Our results suggest that Stat6 and IRS-2 cooperate in promoting both IL-4-induced proliferative and differentiating responses, while an additional signaling mediator that depends on protein tyrosine phosphatase activity contributes to the antiapoptotic activities of IL-4 in primary T cells.     

Fyn membrane localization is necessary to induce the constitutive tyrosine phosphorylation of Sam68 in the nucleus of T lymphocytes.

A close relationship between Sam68, a tyrosine and proline-rich RNA-binding protein, and Src protein tyrosine kinases (PTK) has already been established, also in T lymphocytes. A constitutive phosphorylation of the molecule has also been documented in various transformed T cells, which probably reflects an increased expression of PTK of the Src family. Using the hybridoma T cell line, T8.1, or Jurkat T cells, we investigated the respective contribution of the two Src kinases Fyn and Lck, expressed in T cells, in this phenomenon. By overexpressing the two proteins, we show that the constitutive phosphorylation of Sam68 in vivo directly correlates with cellular Fyn levels, but not with Lck expression, despite the capacity of the PTK to strongly phosphorylate the molecule in vitro. Overexpressed Fyn is mainly localized at the cell membrane. We find that Sam68 phosphorylation, including in the nuclear fraction in which the molecule is predominantly expressed, is lost with a delocalized Fyn mutant deleted of its N-terminal membrane-anchoring domain. Finally, we demonstrate, using a construct encoding a Sam68 molecule without its nuclear localization signal, that nuclear expression of Sam68 is not required for phosphorylation. We conclude that the constitutive phosphorylation of Sam68 in T cells is a Fyn-dependent process occurring in a cell-membrane compartment from which phospho-Sam68 molecules can thereafter accumulate into the nucleus.     

Resistance to experimental autoimmune encephalomyelitis and impaired T cell priming by dendritic cells in Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 mutant mice

Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is expressed on the surface of CD11c(+) dendritic cells (DCs) and macrophages. In this study, we show that mice that express a mutant form of SHPS-1 lacking most of the cytoplasmic region are resistant to experimental autoimmune encephalomyelitis (EAE) in response to immunization with a peptide derived from myelin oligodendrocyte glycoprotein (MOG (35-55)). The MOG (35-55)-induced proliferation of, and production of IFN-gamma, IL-2, and IL-17, by T cells from immunized SHPS-1 mutant mice were reduced compared with those apparent for wild-type cells. The abilities of splenic DCs from mutant mice to stimulate an allogenic MLR and to prime Ag-specific T cells were reduced. Both IL-12-stimulated and TLR-dependent cytokine production by DCs of mutant mice were also impaired. Finally, SHPS-1 mutant mice were resistant to induction of EAE by adoptive transfer of MOG (35-55)-specific T cells. These results show that SHPS-1 on DCs is essential for priming of naive T cells and the development of EAE. SHPS-1 is thus a potential therapeutic target in inflammatory disorders of the CNS and other autoimmune diseases.